Cytochromes P.450 from bovine adrenocortical mitochondria will be characterized with respect to enzyme activity and physical characteristics. The cytochrome concerned with the side-chain cleavage of cholesterol can be isolated in 3 forms with 16, 8 and 4 apparently identical subunits respectively. These forms will be characterized according to subunit composition, heme and phospholipid composition and enzyme activity. The active form(s) of the enzyme will be determined by gradient centrifugation in a medium containing all the factors necessary for side-chain cleavage including substrate. Fractions containing pregnenolone (the product of cleavage), will be identified and corresponding molecular weight will be determined. The possible role of various subunit combinations in partial reactions will be explored (e.g. side-chain cleavage of 20S-hydroxycholesterol and of 20S,22R- dihydroxycholesterol) and also the form of the enzyme catalyzing cleavage of cholesterol sulfate will be examined. Purification of the cytochrome P.450 responsible for 18-hydroxylation will be undertaken.